identification of mutation in the bemisia tabaci para sodium ......table 2. rate of resistance...
TRANSCRIPT
Egypt. J. Agric. Res., 88 (1), 2010
153
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL GENE ASSOCIATED WITH
RESISTANCE TO PYRETHROIDS 153
Central Agricultural Pesticides Laboratory, ARC, Dokki, Giza
(Manuscript received 18 August 2009)
Abstract
Mechanism of resistance to lambda-cyhalothrin in highly
resistant Bemisia tabaci strain can be explained by the voltage-
gated sodium channel which is the primary target site of pyrethroid
insecticides. The super knockdown resistance (super-kdr) to
pyrethroids is caused by changes at specific sites on the para-type
sodium channel protein domain II (IIS4-6). The B. tabaci para-
type sodium channel gene was RT-PCR amplified from lab-strain
(reference), Parent (first generation after treatment with the
insecticide) and generation thirteen after treatment was
considered. The mechanisms of resistance to Lambda-cyhalothrin
(Karat 20% EC) in a Q biotype, highly resistant Bemisia tabaci
strain. Analysis of the sequence of the IIS4-IIS6 region of the para
sodium channel gene of lab-strain, Parent and resistant strain
(G13) revealed two amino acid replacements compared to that of
the SUD-S susceptible strain. One is the leucine to isoleucine
substitution at position 925 (L925I) and allele r1-Q1 (GenBank
accession no. DQ205206) has identical intron sequences with
samples of parent and lab-strain, other is a novel kdr resistant
mutation for B. tabaci, a threonine to valine substitution at position
(T929V) and alleles r2-Q1 (GenBank accession no DQ205207) has
identical intron sequence with G13..
Keywords: Bemisia tabaci, sodium channel gene, insecticide
resistance, pyrethroids
INTRODUCTION
The whitefly, Bemisia tabaci is one of the most serious and key against pests of
numerous crops worldwide. The ability of B. tabaci to develop resistance to
insecticides after only a few applications makes its control problematic in the long
term (Denholm,I. et al 1998)
For the past thirty years, B.tabaci control in Egypt has been based almost
exclusively on conventional insecticides such as organophosphates, pyrethroids and
carbamates. Little work quantifying resistance in B.tabaci in Egypt has been published,
but it is clear that, by the late 1980s, at least some of these broad-spectrum
insecticides were failing in some situations, the pyrethroid cypermethrin, lost its
efficacy during 1989 and 1990, severe resistance to organophosphate and pyrethroids
in B.tabaci from Egyptian cotton fields were established (Abdallah 1991). More recent
data on insecticide efficacy from field trials on cotton shows that many traditional
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL
GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
154
insecticides are no longer effective (Sobiha et al.,1997) and 10 to 12 insecticide
applications per season are now common in the cotton crop.
Insecticide resistance selection is even more marked on vegetable crops, where
the risk of whitefly mediated virus transmission (particularly in tomatoes) has led
farmers to apply organophosphate and/or pyrethroid insecticides two or three times
per day (Hafez El-Kady & Gregor J.D. 2003). Monitoring high resistance levels to
several insecticide classes in field B. tabaci populations from different governorates in
seasons 2000 and 2001, particularly pyrethroids and organophosphates, resistance
factors to the pyrethroid lambda-cyhalothrin was up to 1161.54 fold (Farghaly 2005).
Resistance to pyrethroids in B.tabaci has been associated with enhanced
detoxification by oxidative and hydrolytic pathways. In addition, target site insensitivity
was recently implicated in B. tabaci pyrethroid resistance, (Morin et al., 2002).
Although the kdr mutation L1014F found in many important agricultural pests,
(Soderlund and Knipple 2003) has not been detected in the sweet potato whitefly, two
mutations in the IIS4-5 linker of B.tabaci sodium channel gene, a methionine to valine
substitution at position 918 (M918V) and a leucine to isoleucine alteration at position
925 (L925I) conferred high levels of fenpropathrin resistance in B. tabaci strains
isolated from Arizona, (Morin et al., 2002). A third sodium channel resistance
mutation, a threonine to valine substitution at position 929 (T929V), was implicated
with pyrethroid resistance, in a highly resistant B. tabaci strain (GRMAL-RP) isolated
from Crete, Carboxylesterase (COE) - and cytochrome P450 dependent
monoxygenase-based detoxification was also implicated in pyrethroid resistance in
GRMAL-RP, (Bass et al., 2004)
In the present study, the level of resistance to lambda-cyhalothrin was
estimated in population of B.tabaci. Subsequent insecticide selection was carried out
to build up resistance, and identification of mutations within the voltage-gated sodium
channel gene. This information is fundamental to an understanding of insecticide
resistance and for the development strategies to reduce the risk for lambda-
cyhalothrin.
MATERIALS AND METHODS
FARGHALY, SAYEDA F., et al., 155
1.1-Insecticide used
Lambda-cyhalothrin (Karat 20% EC) (S) (Z)-(1R)-cis-
(R) (Z)-(1S)-cis-
C C
CH3 CH3
CF3 H
C
H H
O
O
H
CH3 CH3
H
Cl
CCN
OH
C O
O
CN
CH
OC C
CF3 H
Cl
+
1.2-Bemsia tabaci strain
Field strain of B.tabaci was collected from Behera governorate (2000) and
maintained without insecticide selection pressure for more than 8 years in the lab of
Central Agricultural Pesticides Laboratory, department of rearing standard insects.
Resistant-strain derived from the Lab-strain was selected with lambda-cyhalothrin for
13 generations (127.08 fold) as shown in table (2).
1.3-Bioassay
The bioassay method for obtaining concentration-response regressions lines was
described by (Prabhaker et al., 1985) with some modification, attached cotton leaves
were dipped for 5 sec. in 100 ml of the desired concentration of each insecticide and
allowed to dry, the treated leaves were laid on a thin layer of 2% agar in small cage
and then twenty adults were transferred into the cage by an aspirator. Mortality was
recorded 24 and 48 hr after treatment. Six concentrations At least were tested for
each insecticide, each test was replicated on five different. Results were expressed as
percentage mortalities, corrected using (Abbott formula, 1925).
1.4-Selection pressure
The population of the resistant-strain which consisted of many thousands was
subjected to laboratory selection pressure with insecticide, at a level producing 30%
mortality to the adult stage. The level of developing resistance was determined at
generations 3, 6, 9 and 13, Resistance ratio (RR) was determined by dividing the LC50
of the R-strain by the LC50 of lab-strain.
2.1-RT-PCR analysis of B. tabaci sodium channel gene sequences
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL
GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
156
The IIS4-5 region of the B.tabaci para-type sodium channel gene was initially
amplified by reverse transcriptase-mediated PCR (RT-PCR). Total RNA was extracted
from susceptible strain, parent types and generation thirteen (S, P and G13). In each
sample, 100 adults were homogenized according to the manufacturer’s instructions.
One RNA sample was produced for each sample. First strand cDNA was synthesized
from total RNA (3μg) by reverse transcriptase (superscript III, RNase H־, ready togo
RT-PCR kit Pharmacia, Aniasham) with oligodT (20 μM) as a primer, according to the
supplier recommended protocol. The cDNA fragments served as templates for
subsequent PCR amplification using the forward primer kdr-1 (5′
GCCAAATCCTGGCCAACT 3′) and reverse Kdr-4 (5′ GAAATTACTCAGCAACAACGC 3′).
The thermal conditions were : denaturation at 94 ºC for 3 min followed by 35 cycles of
94 ºC for 30 s, 55 ºC for 30 s and 72 ºC for 1 min, and final extension at 72 ºC for 10
min.
2.2-Sequence analysis
The presence or absence of the L925I and T929V mutation in individual B.tabaci
adult was analyzed using the manufacturer recommendations (Ahmadian et al., 2000).
The gene fragments of interest were amplified using the primers Bemisia Na-channel
pyro f biotin (5′ TGGCCAACTTTGAATCTGTT 3′) and Bemisia Pyro r (5′
GCATTCCCATCACAGCAAAA 3′) that amplify a 112 bp fragment comprising the L925I
and the T929V mutations.
2.3-Phylogenetic analysis
To determine the phylogenetic relationship between resistant and susceptible
allel sequences, the sequences were aligned using the (Bioeditver 201) for maximum
likelihood analysis. Maximum likelihood analysis were performed by the branch and
bound search, with the tree bisection and reconnection option (TBR).
RESULTS
3.1- RT-PCR amplification of the ПS4-6 region of the B. tabaci para-type
sodium channel gene.
The ПS4-5 region of the B. tabaci para-type sodium channel gene was RT-PCR
amplified from lab-strain (reference), Parent (first generation after treatment with the
insecticide) and generation thirteen. Alignment of the different cDNA clones revealed
the presence of four mismatches and five different sequences (Table 1 and Fig.1).
Compare with susceptible laboratory strain (Sudan) at position 61, cDNA clones
from both lab and parent had A nucleotide instead of T nucleotide the second
substitutions, T nucleotide instead of A nucleotide at position 63, caused a L925I
substitution (sodium channel numbering follows the housefly para-type sodium
FARGHALY, SAYEDA F., et al., 157
channel protein sequence EMBL X96668), and were designated as r1-Q1 (GenBank
accession no. DQ205206). The L925I mutation was previously identified in a B biotype
strain from Arizona and found the linked to synergized pyrethroid resistance (Morin et
al., 2002). They identified in a Q biotype strain (Alon et al., 2006). The third and
fourth substitution, A to C at position 73 and C to T at position 74, occurred only in
cDNA clones from lab-strain and parent type. These substitutions cause a threonine to
leucine substitution at position 929 (T929L). But 13th generation had G instead of A at
position 73 and T instead of C at position 74. These substitutions cause a threonine to
valin (T929V). An identical mutation was recently associated with pyrethroid resistance
in cat flea (Bass et al., 2004, Alon et al., 2006) which agrees with our results. The
cDNA sequence harbouring the T929V mutation was designated as r2-Q1 (GenBank
accession no. DQ205207). The two wild-type sequences lacking the L925I and T929V
mutations, but differing in the codon usage of V981, were designated as s-B and s-Q
(GenBank accession nos. AJ440727 and DQ205204, respectively). The five identified
cDNA sequence had the kdr mutation L1014Q and L1014K [lab-strain, parent and
generation 13th respectively in ПS4-6 region].
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL
GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
158
Fig. 1. Nucleotide sequence of the IIS4-6 region of B. tabaci para-type sodium channel
gene, amplified from the (susceptible strain, parent type and generation
thirteen). The identified cDNA sequences differed by nucleotide substitutions
at positions 61 (L925I) and 73 (T929V).The position of the kdr (L1014) and
super kdr (M918) mutation sites, found in other insects but not in B.tabaci .
FARGHALY, SAYEDA F., et al., 159
Table 1. Nucleotide variation in resistant generation parent and susceptible lab-strain of B.tabaci para-type sodium channel alleles. The sequences span last 145
bp of the first exon and the complete sequence of the first intron in the ПS4-6 region
3.2-The role of L925I and T929V in resistance
To examine the association between the L925I and T929V mutations and
resistance to pyrethroids, bioassay of different generations of resistant strain: G3, G6,
G9 and G13 was carried out (Table 2).
Table 2. Rate of resistance development for Lambda-cyhalothrin in whitefly B.tabaci during selection for 13 generations.
RR* (Resistance ratio) = LC50 of the resistant strain / LC50 of Lab-strain
Generation
61 63 73-74 91 115 116 185 186 244 245 247 248 250
DQ205207.1
DQ205201.1
DQ205206.1
Sample 1
Sample 2
Sample 3
t a gt a g g t t a a a t g
a - ac - - - - - - - - a -
a - ac - - - - - - - - - -
a t ct g t t c c c t t g a
a t ct g t t c c c t t g a
t a gt a g g t t a a a t g
252 253 254 255 259 260 317 318 327 328
DQ205207.1
DQ205201.1
DQ205206.1
Sample 1
Sample 2
Sample 3
a t g a t g t t a a
- a t g - t - - - -
- - - - - - - - - -
t g a t g a a c t c
t g a t g a a c t c
a t g a t g t t a a
strain Karate selected 13 generations strain
Slope ± EC LC50ppm(95%FL) RR*
Lab.strain 1.39 ± 0.43 6.86 (3.43-11.39) -----
Parent 1.12 ± 0.44 5.36 (1.77-13.53 0.78
G3 2.86 ± 0.82 21.10 (---------) 3.10
G6 1.63 ± 0.24 30.63 (23.78-41.28) 4.46
G9 1.12 ± 0.37 98.94 (27.02-215.42) 14.42
G13 1.45 ± 0.19 127.08 (97.02-175.51 18.52
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL
GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
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Table 3. Mutation found in the transmembrane segment IIS4 of the para sodium channel gene in different strains of B. tabaci
a Numbering of amino acid residues follows translation of Musca domestica sodium channel
gene (X96668).
b Sequences reported by Morin et al.,( 2002 )
c This study
d Sequences reported by Emmanouil et al.,( 2006 )
e Replacements occurred in single haplotypes
which has the r2-Q1 carrying the T929V mutations), parent and lab-strain which has
both the r1-Q1 sequences (carrying L925I mutations) (Table 3) and extracted RNA
from lab-strain (s), parent (p) and three generations of resistant strain(G4, G9 and G13)
which appeared bands with M.W. 399, 417, 416, 418 and 420 KDa respectively. (Table
4 and fig. 2)
The role of L925I has been well documented in B. tabaci pyrethroid resistance
(Morin et al., 2002), the second mutation, threonine (ACT)-valine (GIT) replacement
at amino acid 929 (T929V) identified in the G13 pyrethroid resistant strain (Table 3), is
the same kdr resistance mutation previously reported in the cat flea Centocephalids
felis (Bass et al., 2004) and pyrethroid resistance from Crete in the B. tabaci (
Roditakis et al., 2006) the role of the T929V in pyrethroid resistance has been
established by site directed mutagenesis and functional expression studies in
Drosophila (Atkinson, 2002).
Strain Description Sequence at position
925a
Sequence at
position 929a
SUD-Sb Lab susceptible (Sudan) TTA Leucine ACT threonine
Sample1
Parentc
Pyrethroid resistant lab strain 1st
generation
ATT isoleucine CTT leucine
Sample2
susceptiblec
Susceptible lab strain ATT isoleucine CTT leucine
Sample3
Generation13c
Pyrethroid resistant lab strain
13th generation
TTA Leucine GTTd Valine
GRMAL-RPd Pyrethroid resistant from Crete ATAe Isoleucinee GTTd Valine
FARGHALY, SAYEDA F., et al., 161
Fig. 2. Gel analysis of RT-PCR amplification using Kdr-1 forward and Kdr-4 reverse.
The wild-type allele was amplified from three generations. DNA was
extracted from 100 whitefly laboratory-strain (s), three generations of
resistant strain (G4, G9, G13). The position of the amplified PCR fragment
was (370 bp)
Table 4 . Molecular weight of RNA bands for different samples of resistant strain
3.4- Nucleotide variation in the first axon and first intron of domain ПS4-6
In the Q-biotype, it is found that two resistant alleles r2-Q1 (GenBank accession
nos. DQ205207) has identical intron sequence with G13. The overall nucleotide
variation was 0.000. and allele r1-Q1 (GenBank accession no. DQ205206) has identical
intron sequences with samples of parent and lab-strain. The overall nucleotide
variation for samples was 0.000 but the samples differs from r1-Q1 by 22 nucleotide
substitutions (table 1). The overall nucleotide variation for samples with r1-Q1
(GenBank accession no. DQ205206) was 0.066.
Generation Molecular weight
Lab. strain
Parent
Generation 4
Generation 9
Generation 13
399
417
416
418
420
IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL
GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
162
3.5- Phylogenetic analysis
Phylogenetic analysis using maximum likelihood is shown in (Table 5 and Fig
3). The value of similarity between G13 and r2-Q1 (GenBank accession no. DQ205207)
was 100.00. While the value of similarity for sample1, sample2 and r1-Q (GenBank
accession no. DQ205206) was 93.38% because the two samples differs from allele r1-
Q by 22 nucleotide substitution .(Table 1).
Fig 3. Maximum likelihood analysis of resistant and susceptible allele Sequences
produced from the first exon (145bp) and first intron in ( 730bp) the IIS4-6
domain of the B.tabaci para-sodium channel gene. Analyses were performed
by the branch and bound search, with the tree bisection and reconnection
option (TBR).
Table 5. Estimated similarity for allele sequences produced from the first exon (145 bp) and first intron ( 730 bp) in the IIS4-6 domain of the B. tabaci para-
type sodium channel gene.
DQ205207 DQ205201 DQ205206 Sample1 Sample2 Sample3
DQ205207 100.00 ------- -------- ------- ------- ------
DQ205201 67.10 100.00 -------- ------- ------- ------
DQ205206 99.18 68.45 100.00 ------- ------- ------
Sample1 93.38 59.42 93.38 100.00 ------- ------
Sample2 93.38 59.42 93.38 100.00 100.00 ------
Sample3 100.00 67.10 99.18 93.38 93.38 100.00
FARGHALY, SAYEDA F., et al., 163
DISCUSSION
Point mutations in the insect para sodium channel gene that reduce the
sensitivity to pyrethroids have been identified in several insect species. Most studies
have focused on the IIS4-5 region, using degenerate primers designed from
conserved sequences in these membrane-spanning segments (Martinez-Torres et al.,
1997). Using this strategy, we amplified from B. tabaci (a resistant lab strain from
Egypt) the cDNA fragment spanning the II4-6 region. Through cloning and sequencing
we identified two mutations, (L925I and T929V). The two mutations have been
previously reported in the GRMAL-RP (a resistant lab strain from Crete) of B. tabaci
(Roditakis et al., 2006). The L925I mutation was reported by (Morin et al., 2002),
while functional expression studies in oocysts showed that it is able to reduce channel
sensitivity to deltamethrin by over 2000-fold (Atkinson 2002). In contrast, the
mutation at position 918 (methionine), which was associated with pyrethroid
resistance in a laboratory selected whitefly strain [but not in pyrethroid-resistant field-
collections from Arizona (Morin et al., 2002)] was not also found in GRMAL-RP (a
resistant lab- strain from Crete) (Emmanouil et al., 2006), which identical with this
investigation in pyrethroid resistant-lab strain from Egypt.
The level of resistance conferred by each of them is difficult to infer. We are
currently undertaking experiments to separate the two mutations, as well as to obtain
susceptible whiteflies of similar genetic background, to evaluate the exact level of the
resistance conferred by each mutation and their impact (if any) on fitness. As in many
agricultural pests, showing that whiteflies which carry the V929 (or I959) alone are
viable, in line with previous studies (Morin et al., 2002 and Bass et al., 2004).
Horowitz et al.,(2003) suggested that although B-type may survive better than
Q-type B. tabaci under untreated conditions, the faster occurrence and development
of resistance in Q biotype favours its predominance in areas with heavy insecticide
pressure history. The classification of the highly resistant strains (field strain and lab
resistant) whiteflies isolated from Behera Governorate as Q biotype, indicates that the
use of insecticides may have increased the occurrence of these biotype in Egypt.
However further studies are needed to explain the B and Q biotypes genetically
isolation. Nevertheless, our data show little or no gene flow between B and Q biotypes
of B. tabaci since the introduction of the widespread use of synthetic insecticides that
target the para-type voltage gated sodium channel such as pyrethroids.
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II S 4 - 6 )
RT-PCR )
925L925 I
r1-Q1DQ205206
929T 929 V )r2-Q1
DQ205207